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, 288 (17), 12253-68

Epithelial Cell Adhesion Molecule (EpCAM) Regulates Claudin Dynamics and Tight Junctions

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Epithelial Cell Adhesion Molecule (EpCAM) Regulates Claudin Dynamics and Tight Junctions

Chuan-Jin Wu et al. J Biol Chem.

Abstract

Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins.

Figures

FIGURE 1.
FIGURE 1.
EpCAM localizes to lateral interfaces of human intestinal epithelial cells below tight junctions. A, human carcinoma (T84) cells growing as colonies on coverslips were stained with anti-EpCAM and examined using confocal laser immunofluorescence microscopy. B and C, post-confluent T84 cell monolayers growing on membranes in Transwell chambers were fixed, and double-stained with anti-EpCAM and anti-occludin (B, XZ image) or anti-EpCAM and anti-ZO-1 (C, three-dimensional image). D, lightly fixed frozen sections of human small intestine were stained with anti-EpCAM and anti-occludin and analyzed via confocal microscopy. Scale bars, 10 μm (A–C), or 20 μm (D).
FIGURE 2.
FIGURE 2.
Reduction of EpCAM expression changes the morphology and physiology of colon cancer cells. T84 and Caco-2 cells were infected with retroviruses expressing control or EpCAM shRNAs, and stable transductants were selected using puromycin. A, cell lysates from transduced T84 (upper panel) or Caco-2 (lower panel) cells were assessed for EpCAM content using Western blotting. B, phase contrast images reveal morphologies of unmanipulated as well as control vector- or EpCAM shRNA-transduced T84 cells. Cells were plated in 10-cm dishes and cultured for 6 days prior to imaging. C and D, control vector- or EpCAM shRNA-transduced T84 (C) or Caco-2 (D) cells were assessed for proliferative activity using the WST-1 assay. Bars represent daily A450 determinants (means ± S.D.) for each cell line normalized to the day 1 A450 for that cell line. E, abilities of control vector-transduced and EpCAM shRNA 2-expressing Caco-2 cells to migrate were studied using a wound healing assay. Defects in monolayers were created by adding cells into 6-well plates containing inserts in each well. After incubation for 24 h in medium containing 0.1% FBS, inserts were removed, and phase contrast photomicrographs were obtained at the indicated times. Representative images from n = 3 experiments are shown. Residual wound areas (means ± S.E.) after 16 h were determined using ImageJ. Scale bars, 200 μm. *, p < 0.05; **, p < 0.01.
FIGURE 3.
FIGURE 3.
Regulation of TEER and tight junction stability by EpCAM. A and B, monolayers of control vector- or EpCAM shRNA-transduced T84 (A) or Caco-2 (B) cells were cultured in Transwells, and TEERs were determined daily for T84 or every other day for Caco-2 cells. Mean TEERs ± S.E. are depicted. Representative results from one of three experiments are shown. C, T84 cell monolayers were grown and monitored for acquisition of TEER. Maximal TEERs were obtained after 9 days of incubation, and monolayers were subsequently incubated in EGTA-containing (calcium-depleted) or regular medium for 40 min, fixed, and stained with anti-occludin. D, digital images corresponding to 10 random fields of anti-occludin-stained retrovirus-transduced T84 monolayers that had been calcium-depleted were subjected to quantitative analysis using AngioTool. Aggregate lengths of tight junction networks and numbers of network junctions per field (means ± S.E.) are shown. Representative results from one of three experiments are presented. Scale bars, 10 μm (C, upper panel) or 20 μm (C, lower panel). *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
EpCAM depletion does not markedly alter the distribution of adherens junction- or desmosome-associated proteins or epithelial polarity. Stable control vector- or shEpCAM 2-transduced T84 cells were grown in Transwells for 8–10 days to allow development of maximal TEER and then fixed and stained for ZO-1 and E-cadherin (E-cad) (A), occludin (Occl) and β-catenin (β-cat) (B), ZO-1 and desmoglein 2 (Dsg2) (C), for Na/K-ATPase (red) and CD26 (green) (D), or for myosin IIA (red) and EpCAM (green) (E). Results shown are representative of those observed in three experiments. Scale bars, 10 μm (A–C, left panels, and D and E, XZ images), or 20 μm (right panels, XY images).
FIGURE 5.
FIGURE 5.
EpCAM interacts with claudin-7 and claudin-1. A, control vector-transduced or EpCAM knockdown T84 cells were solubilized in Triton X-100-containing buffer, and lysates were immunoprecipitated (IP) with control IgG or anti-EpCAM Ab. Immunoprecipitates were resolved via SDS-PAGE, and gels were stained with Coomassie Blue. Differentially represented bands/proteins were characterized using mass spectrometry. B and C, immunoprecipitates from vector-transduced or EpCAM knockdown T84 cells (B) or Caco-2 cells (C) acquired as in A were fractionated using SDS-PAGE and immunoblotted with anti-claudin-7, -1, -2 or -4 or anti-occludin Abs. D–F, co-localization of EpCAM with claudin-7 and claudin-1 detected using confocal scanning laser microscopy. D, T84 cells were grown on coverslips for 2 days, fixed with paraformaldehyde, and permeabilized with 0.5% Triton X-100. E, T84 monolayers were cultured on Transwell filters for 8 days, fixed with cold acetone/ethanol (3:1), and stained with anti-EpCAM and anti-claudin-7 Abs. A mid-level optical section (below TJ) is displayed. F, Caco-2 monolayers were grown on Transwell filters for 23 days, fixed with cold methanol, and stained for EpCAM and claudin-1. A mid-level optical section (below TJ) is displayed. Scale bars, 20 μm.
FIGURE 6.
FIGURE 6.
Claudin-7 mediates EpCAM-claudin-1 interactions by associating with claudin-1. A, T84 cells transfected with negative control (siNeg) or claudin-7 siRNA (siC7) were solubilized, and cell lysates were immunoprecipitated (IP) with control IgG or anti-EpCAM Ab as in Fig. 5A. Immunoprecipitates and cell lysates were fractionated using SDS-PAGE and immunoblotted with anti-claudin-1, anti-claudin-7, or anti-EpCAM. B, anti-claudin-1 immunoprecipitates of T84 cell lysates acquired as in A were fractionated using SDS-PAGE and immunoblotted with anti-claudin-7 or anti-claudin-1. C, COS-7 cells were co-transfected with pTRIP-claudin-1 and pcDNA3-claudin-7HA or control empty vectors (Vec) using Lipofectamine. Cells were lysed in Triton X-100-containing buffer 24 h after transfection, and cell lysates were immunoprecipitated with anti-HA antibody. SDS-PAGE-resolved immunoprecipitates were blotted with anti-claudin-1 or anti-claudin-7.
FIGURE 7.
FIGURE 7.
EpCAM protects claudin-7 and claudin-1 from degradation via a lysosome-dependent pathway. A, shVector- or shEpCAM-transduced T84 cells were lysed in Triton X-100-containing buffer, and normalized amounts of solubilized proteins were quantified by immunoblotting (IB). B, shVector- or shEpCAM-transduced T84 cells or Caco-2 cells were lysed with RIPA buffer, and supernatants were cleared via centrifugation at 12,000 × g for 15 min. Normalized amounts of cell lysate proteins were resolved using SDS-PAGE and immunoblotted for the indicated proteins. β-Actin was used as a loading control. C, T84 cell total RNA was analyzed for claudin-7 and claudin-1 mRNA content using real time RT-PCR. Claudin mRNA levels in each sample are expressed relative to GAPDH mRNA levels and were normalized such that expression levels in control (vector-transduced) cells = 1. D and E, T84 cells were plated for 40 h and subsequently treated with the lysosome inhibitor chloroquine for 24 h (D) or the proteasome inhibitor lactacystin for 20 h (E) (or appropriate diluent controls) at the indicated concentrations, and RIPA buffer lysates were prepared. Cell lysates were normalized for protein concentrations; lysate proteins were resolved via SDS-PAGE, and EpCAM, claudin-7, and claudin-1 were detected via immunoblotting. β-Actin was used as a loading control. F, vector-transduced control and EpCAM shRNA 2-expressing T84 cells were treated with, or without, 100 μm chloroquine for 24 h and lysed with RIPA buffer. Cell lysates were normalized for protein concentrations and immunoprecipitated with anti-claudin-7 antibody. Immunoprecipitates were blotted with anti-ubiquitin (Ub) and anti-claudin-7.
FIGURE 8.
FIGURE 8.
Intracellular claudin-7, claudin-1, and EpCAM localize to endosomal/lysosomal compartments. A and B, Caco-2 cells grown on chamber slides were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and co-stained for claudin-1 and Lamp1 (A) or EpCAM and Lamp1 (B). C, stable shVector- or shEpCAM-transduced Caco-2 cells were treated with or without 100 μm chloroquine for 24 h and fixed with 4% paraformaldehyde. Fixed cells were permeabilized with 0.5% Triton and co-stained with anti-claudin-7 and anti-Lamp1. Stained cells were imaged using confocal laser immunofluorescence microscopy. Scale bars, 50 μm.
FIGURE 9.
FIGURE 9.
Reduction of EpCAM levels promotes accumulation of claudin-7 and claudin-1 in tight junctions. A and B, shVector- or shEpCAM-transduced T84 cell monolayers were grown in Transwells for 9 days to allow acquisition of maximal TEER, fixed with cold acetone/ethanol (3:1), stained for EpCAM and claudin-7 (A), ZO-1 and claudin-1 (B), or claudin-4 and claudin-5 (C) and analyzed using confocal microscopy (XZ images). D, ZO-1 and claudin-7 distributions were assessed in T84 cells using an analogous approach (en face images). Each panel depicts representative data from three to four experiments. Scale bars, 10 μm (A–C) or 20 μm (D). E, shVector- or shEpCAM-transduced T84 cell monolayers grown in Transwells were fixed and stained for ZO-1 and claudin-1 or claudin-7 as in B and D. Ten randomly selected XZ images as shown in B corresponding to each condition were analyzed to determine co-localization of claudin-7 (upper panel) or claudin-1 (lower panel) with ZO-1 signals. Co-localization coefficients were shown. **, p < 0.01. F, Caco-2 cell monolayers with maximal TEER acquired after culturing for 23 days were fixed with cold methanol and then stained for claudin-1 and ZO-1 (en face images). Representative data from one of three experiments are shown. Scale bar, 10 μm.
FIGURE 10.
FIGURE 10.
Regulation of claudin levels by EpCAM requires physical interactions involving EpCAM and claudins, and claudin redistribution is responsible for the modulation of TJ function by EpCAM. A, T84 cell clone that had markedly reduced EpCAM expression after transduction with shEpCAM 2 was transfected with pcDNA3 or pcDNA3 containing HA-tagged EpCAM or EpCAM(A279IG283I) (EpCAMmut) using electroporation. Cells were lysed in Triton X-100-containing buffer 48 h after transfection, and cell lysates that had been normalized for protein content were immunoprecipitated with anti-HA antibody. SDS-PAGE-resolved immunoprecipitates were blotted with anti-claudin-7, anti-claudin-1, and anti-EpCAM. B, cloned EpCAM knockdown T84 cells were transfected as in A with pcDNA3 or pcDNA3 containing HA-tagged EpCAM or EpCAM(A279IG283I). Cell lysates normalized for protein content were obtained 48 h after transfection, and SDS-PAGE-resolved proteins were immunoblotted with anti-claudin-7 and anti-EpCAM. C, Caco-2 clone with dramatically reduced EpCAM expression subsequent to transduction with shEpCAM 2 was transfected with pcDNA3 or plasmid encoding HA-tagged EpCAM or EpCAM(A279IG283I). After selection with G418 for several weeks, cells with EpCAM expression comparable with the endogenous levels were isolated via preparative flow cytometry. Sorted cells were plated into Transwells and cultured for 21 days and stained for HA (EpCAM) and claudin-1 after fixation with cold methanol. Scale bar, 10 μm. D and E, stable shVector- or shEpCAM 2-transduced Caco-2 cells were transfected with control siRNA (siNeg) or claudin-7 siRNA and claudin-1 siRNA (siC7C1) duplexes using electroporation. Twenty four h later, 5 × 105 cells were plated into Transwell chambers with 12-mm diameter polyester filters, and TEERs were determined daily thereafter. RIPA buffer cell lysates were collected over the course of the experiment and examined for claudin-7 and claudin-1 expression using Western blotting (D). Mean TEERs ± S.E. are depicted (E). **, p < 0.01, compared with Caco-2/shEpCAM 2 cells transfected with siC7C1 or compared with Caco-2/shVector cells transfected with siNeg. Representative results from one of three experiments are shown. Vec, vector.

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